Potential Novel Role of Membrane-Associated Carbonic Anhydrases in the Kidney

Carbonic anhydrases (CAs), because they catalyze the interconversion of carbon dioxide (CO2) and water into bicarbonate (HCO3−) and protons (H+), thereby influencing pH, are near the core of virtually all physiological processes in the body. In the kidneys, soluble and membrane-associated CAs and their synergy with acid–base transporters play important roles in urinary acid secretion, the largest component of which is the reabsorption of HCO3− in specific nephron segments. Among these transporters are the Na+-coupled HCO3− transporters (NCBTs) and the Cl−-HCO3− exchangers (AEs)—members of the “solute-linked carrier” 4 (SLC4) family. All of these transporters have traditionally been regarded as “HCO3−“ transporters. However, recently our group has demonstrated that two of the NCBTs carry CO32− rather than HCO3− and has hypothesized that all NCBTs follow suit. In this review, we examine current knowledge on the role of CAs and “HCO3−” transporters of the SLC4 family in renal acid–base physiology and discuss how our recent findings impact renal acid secretion, including HCO3− reabsorption. Traditionally, investigators have associated CAs with producing or consuming solutes (CO2, HCO3−, and H+) and thus ensuring their efficient transport across cell membranes. In the case of CO32− transport by NCBTs, however, we hypothesize that the role of membrane-associated CAs is not the appreciable production or consumption of substrates but the minimization of pH changes in nanodomains near the membrane.


Introduction
Carbonic anhydrases (CAs) are ubiquitous metalloenzymes that play a fundamental role in numerous vital processes, including cellular metabolism, carbon dioxide (CO 2 ) and ion transport, and acid-base homeostasis [1][2][3][4][5]. These enzymes are important because they permit rapid interconversion of CO 2 and bicarbonate (HCO 3 − ), thereby promoting the rapid buffering of acid or alkali loads and facilitating-to various degrees-the diffusion of CO 2 , HCO 3 − , H + , and other buffers [5]. For example, in red blood cells (RBCs), intracellular CA provides a mechanism for the efficient carriage of CO 2 from peripheral tissues to the lungs [5]. In the kidneys, as we shall see later, the synergistic action of soluble and membrane-associated CA activity permits efficient reabsorption of filtered HCO 3 − . CAs are also linked to a variety of pathological processes, including renal tubular acidosis, osteopetrosis, osteoporosis, and tumor proliferation [6][7][8][9][10][11][12]. Inhibition of CAs has important pharmacological implications, for example, in the treatment of glaucoma, epilepsy, acute mountain sickness, and, more recently, even cancer [13][14][15][16][17]. CAs may also be useful as markers for cancer diagnosis [17][18][19].
Because of their involvement in physiological and pathological processes, these enzymes continue to be the focus of current investigations. CA was discovered in 1933 in Because of their involvement in physiological and pathological processes, these enzymes continue to be the focus of current investigations. CA was discovered in 1933 in bovine RBCs by Meldrum and Roughton during their study of the interactions between CO2 and HCO3 − in blood [20]. The first three isozymes to be identified were CA A (later renamed CA III), CA B (later CA I), and CA C (later CA II) [21][22][23][24]. We now recognize eight evolutionally unrelated families of CAs: -, β-, γ-, δ-, ζ-, η-, θ-, and ι-CAs [2,13,[25][26][27]. α-CAs, which include CAs I-III, are found in numerous eukaryotic and prokaryotic organisms and are the most widely studied and characterized because of their importance in higher animals [28,29]. β-CAs are found in both eukaryotes (e.g., algae, plant chloroplast, and fungi) and prokaryotes. Recently, β-CAs have also been identified in animals, particularly in invertebrates [28][29][30][31]. γ-CAs are mainly found in archaea and some bacteria, and δ-and ζ-CAs are expressed in some marine diatoms [28,29,32]. The η-CAs are found in the protozoan Plasmodium species, which cause malaria in humans [33]. In the past few years, investigators have identified θ- [34] and ι-CAs [25] in marine diatoms.
In this review, we focus exclusively on the -family, which predominates among animals. The α-CA family comprises thirteen enzymatically active isozymes and three inactive ones. Figure 1 shows their different sub-cellular localizations (i.e., cytosolic, mitochondrial, secretory, and membrane-associated). Each member of the -CA family has a distinct combination of kinetic properties and tissue-specific distribution. For example, CA II and CA III are both found in the cytosol but have the highest and lowest catalytic activities, respectively [39][40][41]. CA II is widely expressed throughout the body, whereas CA III is expressed mainly in muscle cells [2]. Moreover, members of the α-CA family have different susceptibilities to inhibitors. For example, CA II can be easily blocked by acetazolamide (ACZ) and topimirate (i.e., very small amounts of inhibitor can block enzyme activity). CA IV can be blocked more easily by ACZ than topimirate [42]. Cartoon illustrating the sub-cellular localization of the currently known α-CA family members. According to their sub-cellular localization, -CAs can be classified into four groups: cytosolic (CA I, CA II, CA III, CA VII, CA VIII, CA X, CA XI, and CA XIII), mitochondrial (CA VA and CA VB), secretory (CA VI), and membrane-associated (CA IV, CA IX, CA XII, CA XIV, and CA XV). The cytosolic isozymes are all active (indicated in dark green) except for CA VIII, X, and XI (in grey), which are also called CA-related proteins (CARPs). Of the membrane-associated CAs, CA IV and CA XV are glycosylphosphatidylinositol (GPI)-linked, whereas CA IX, CA XII, and CA XIV are transmembrane. Note that the mitochondrial CA VA and CA VB are encoded by two different genes [43,44]. Cartoon illustrating the sub-cellular localization of the currently known α-CA family members. According to their sub-cellular localization, α-CAs can be classified into four groups: cytosolic (CA I, CA II, CA III, CA VII, CA VIII, CA X, CA XI, and CA XIII), mitochondrial (CA VA and CA VB), secretory (CA VI), and membrane-associated (CA IV, CA IX, CA XII, CA XIV, and CA XV). The cytosolic isozymes are all active (indicated in dark green) except for CA VIII, X, and XI (in grey), which are also called CA-related proteins (CARPs). Of the membrane-associated CAs, CA IV and CA XV are glycosylphosphatidylinositol (GPI)-linked, whereas CA IX, CA XII, and CA XIV are transmembrane. Note that the mitochondrial CA VA and CA VB are encoded by two different genes [43,44].
In the following sections, we examine the role of CA isozymes in renal acid-base physiology by reviewing established knowledge as well as new insights obtained by recent work from our group on the substrate identity of "HCO 3 − " transporters of the "solute-linked carrier 4" (SLC4) family. After a brief overview of the members of the SLC4 family that play a role in renal acid-base physiology, we systematically review CA expression, sub-cellular localization, and functional interaction with SLC4 family proteins along the nephron segments that engage in acid secretion, including HCO 3 − reabsorption. We conclude by suggesting that a role of membrane-associated CAs may be to reduce local pH changes caused by the transmembrane movement of carbonate (CO 3 2− ) via the Na + -coupled HCO 3 − transporter (NCBT) subfamily of SLC4 rather than to produce the substrate(s) for acid-base transport by NCBTs, as traditionally thought.

Catalytic Mechanism of α-Carbonic Anhydrases
In the absence of CAs, the interconversion of CO 2 and HCO 3 − is a two-step bidirectional process: In the first of the two sequential reactions (each governed by its own pK), CO 2 hydration describes the combination of CO 2 with water (H 2 O) to form carbonic acid (H 2 CO 3 ). Dehydration describes the opposite direction. In the second reaction, H 2 CO 3 dissociates into HCO 3 − and H + ; this reaction, too, is reversible. The two-step interconversion of CO 2 and HCO 3 − is extremely slow because of the high activation energy of the first of the two reactions. A CA enzyme (activator) lowers the overall activation energy by bypassing the H 2 CO 3 intermediate as the CA catalyzes the thermodynamically equivalent reaction: (2) Figure 2 shows the catalytic mechanism of reaction (2) for all α-CAs. Known as the zinc-hydroxide mechanism [45,46], the central catalytic step-when viewed in the forward direction in reaction (2)-is the reaction of CO 2 with a zinc-hydroxide (i.e., Zn 2+ -OH − ). The presence of the hydroxyl group rather than H 2 O (i.e., Zn 2+ -H 2 O, a less strong nucleophile than Zn 2+ -OH − ) is necessary for the enzyme to be active and able to start the forward cycle [47].
In the state illustrated in the upper left of Figure 2, the Zn 2+ of the α-CAs coordinates with three conserved histidine (His) residues and one hydroxyl group. Because in biological enzymes, Zn 2+ can have up to six coordination sites, when α-CAs are in the Zn 2+ -OH − state, the Zn 2+ has two free coordination sites [48,49]. In step #1 of the forward reaction cycle, the nucleophile O − (red-colored) in the hydroxyl group performs a nucleophilic attack on the carbon of CO 2 (green-colored), generating HCO 3 − on the active site. An electron-rich O − (green-colored) on the HCO 3 − molecule makes a fifth coordination with Zn 2+ . In step #2, H 2 O interacts with Zn 2+ via its O atom (blue-colored). This sixth coordination to Zn 2+ destabilizes the two bonds between Zn 2+ and HCO 3 − so that, in step #3, HCO 3 − dissociates from the enzyme active site. In step #4, Zn 2+ acts as a Lewis acid, promoting deprotonation of the coordinated H 2 O, thereby releasing H + (blue-colored) and regenerating the Zn 2+ -OH − (we now morph the color from blue to red to restart the cycle) form of the enzyme [50].
The presence of different amino acids-with different positions and orientations-near the active site provides different catalytic activity and kinetics to the various CA isozymes. For example, the presence of histidine at position 64 (His-64) of CA II favors H 2 O deprotonation (i.e., step #4 in Figure 2) by transferring H + to the buffer in the media. Indeed, sitedirected mutagenesis studies have shown that replacing this "shuttle histidine" with either alanine or glutamine inhibits CA II catalytic activity [51,52]. In the case of CA III-which has a structure very similar to that of CA II but much lower catalytic activity-substitution of the naturally occurring lysine-64 (in CA III) with His-64 enhances its enzymatic activity [39]. In addition, the presence of bulky amino acids next to His-64 in CA II, or 4 of 25 equivalently next to His-88 in CA IV, can also decrease catalytic activity. Other residues can modulate catalytic activity. Among them, threonine (Thr) 199 in CA II is important for stabilizing HCO 3 − in the active site by forming a network of hydrogen bonds [53,54].
high activation energy of the first of the two reactions. A CA enzyme (activator) lowers the overall activation energy by bypassing the H2CO3 intermediate as the CA catalyzes the thermodynamically equivalent reaction: CO2 + H2O ⇌ HCO3 − + H + .
(2) Figure 2 shows the catalytic mechanism of reaction (2) for all -CAs. Known as the zinc-hydroxide mechanism [45,46], the central catalytic step-when viewed in the forward direction in reaction (2)-is the reaction of CO2 with a zinc-hydroxide (i.e., Zn 2+ -OH − ). The presence of the hydroxyl group rather than H2O (i.e., Zn 2+ -H2O, a less strong nucleophile than Zn 2+ -OH − ) is necessary for the enzyme to be active and able to start the forward cycle [47]. Figure 2. Schematics of the catalytic mechanism of action of α-CAs. In step #1, CO2 (green-colored) reacts with a zinc-bound hydroxyl (OH − , red-colored), generating a coordinated HCO3 − . In step #2, (2) (4) Figure 2. Schematics of the catalytic mechanism of action of α-CAs. In step #1, CO 2 (green-colored) reacts with a zinc-bound hydroxyl (OH − , red-colored), generating a coordinated HCO 3 − . In step #2, The α-CA family members are all enzymatically active except for the three cytosolic forms CA VIII, X, and XI that, for lack of one or more of the three conserved histidine residues in the active site, are unable to perform the cycle illustrated in Figure 2. These three CAs are known as CA-related proteins (CARPs). Despite being apparently linked to various diseases, their physiological role is not well understood [55]. An intriguing possibility is that, like the CA-like domain of the receptor protein tyrosine phosphatase γ (see below), the CARPs may be molecular sensors of CO 2 and/or HCO 3 − .

Carbonic Anhydrases in Nanodomains Adjacent to the Cell Membrane
In addition to reactions (1) and (2) above, the chemistry of HCO 3 − also includes the dissociation of this species into carbonate (CO 3 2− ) and H + : In contrast to reaction (2), reaction (3) is very fast and does not rely on the action of a catalyst. Another difference is their respective pK values,~6.1 at 37 • C for reaction (2) and 10.3 for reaction (3). As a consequence, reaction (2) is dominant at physiological pH, and the majority of CO 2 -related carbon species in the body is in the form of HCO 3 − . It is informative to examine how the transmembrane movements of CO 2 , HCO 3 − , H + , and CO 3 2− as well as how membrane-associated CAs (all of which are on the outer surface of the plasma membrane; see Figure 1) affect the chemical equilibria of reactions (2) and (3).
When CO 2 , HCO 3 − , H + , or CO 3 2− move across a cell membrane, they perturb the chemical equilibria of reactions (2) and (3) in the nanodomain near the outer surface (oS) of the membrane, as illustrated in Figure 3. Comparable, though opposite, reactions occur at the inner surface of the membrane (not shown). In panels A-D, we orient all fluxes in the direction that would produce a fall in pH oS . If CO 2 moves out of the cell ( Figure 3A), CA tends to reestablish the chemical equilibrium of reaction (2)  inner surface of the membrane (not shown). In panels A-D, we orient all fluxes in the direction that would produce a fall in pHoS. If CO2 moves out of the cell ( Figure 3A), CA tends to reestablish the chemical equilibrium of reaction (2) near the outer surface by consuming the exiting CO2 (i.e., minimizing the rise in [CO2]oS) to produce HCO3 − and H + (i.e., accentuating the rise in [H + ]oS/fall in pHoS). If CO2 were entering the cell, the CA would do just the opposite. Thus, in both situations (i.e., independently of the direction of CO2 movement), CA tends to minimize changes in [CO2]oS but maximize changes in pHoS. If H + moves out of the cell ( Figure 3C), CA tends to consume H + (i.e., minimizing the rise in [H + ]oS) as well as HCO3 − (i.e., accentuating the fall in [HCO3 − ]oS). If H + were entering the cell, the CA would do just the opposite. Thus, independently of the direction of H + movement, the CA tends to minimize changes in pHoS.
A more complex scenario occurs when CO3 2− moves across the cell membrane because CO3 2− movement first produces a major perturbance in the chemical equilibrium of reaction (3) near the outer surface of the membrane, followed by a large secondary effect on the equilibrium of reaction (2) because [HCO3 − ]oS >> [CO3 2− ]oS under physiological conditions. If CO3 2− enters the cell ( Figure 3D), reaction (3) tends to replenish the lost CO3 2− , thereby producing H + near the outer surface of the membrane. Catalyzing reaction (2), CA will then consume much of the newly formed H + . If CO3 2− were exiting the cell, the CA would do just the opposite. Thus, independently of the direction of CO3 2− movement, the CA tends to minimize not only changes in [CO3 2− ]oS but also changes in pHoS.
In summary, all four panels in Figure 3 show us that CA near the membrane tends to stabilize the concentration of the transported solute, regardless of direction. Based on intuition, one might predict that these actions, in principle, would universally accelerate  If HCO 3 − moves into the cell ( Figure 3B), CA tends to produce HCO 3 − (i.e., minimizing the fall in [HCO 3 − ] oS ) as well as H + (i.e., accentuating the rise in [H + ] oS /fall in pH oS ). If HCO 3 − were exiting the cell, the CA would do just the opposite. Thus, independently of the direction of HCO 3 − movement, the CA tends to minimize changes in [HCO 3 − ] oS but maximize changes in pH oS .
If H + moves out of the cell ( Figure 3C), CA tends to consume H + (i.e., minimizing the rise in [H + ] oS ) as well as HCO 3 − (i.e., accentuating the fall in [HCO 3 − ] oS ). If H + were entering the cell, the CA would do just the opposite. Thus, independently of the direction of H + movement, the CA tends to minimize changes in pH oS .
A more complex scenario occurs when CO 3 2− moves across the cell membrane because CO 3 2− movement first produces a major perturbance in the chemical equilibrium of reaction (3) near the outer surface of the membrane, followed by a large secondary effect on the equilibrium of reaction (2) because [HCO 3 − ] oS >> [CO 3 2− ] oS under physiological conditions. If CO 3 2− enters the cell ( Figure 3D), reaction (3) tends to replenish the lost CO 3 2− , thereby producing H + near the outer surface of the membrane. Catalyzing reaction (2), CA will then consume much of the newly formed H + . If CO 3 2− were exiting the cell, the CA would do just the opposite. Thus, independently of the direction of CO 3 2− movement, the CA tends to minimize not only changes in [CO 3 2− ] oS but also changes in pH oS . In summary, all four panels in Figure 3 show us that CA near the membrane tends to stabilize the concentration of the transported solute, regardless of direction. Based on intuition, one might predict that these actions, in principle, would universally accelerate transport. However, this is not true. In the case of CO 2 ( Figure 3A), the work from our group [56][57][58]which extends the earlier work of Gutknecht and colleagues [59]-shows that the stimulation of CO 2 transport by CAs is quite large. The reason is that the CA-catalyzed reaction can have very large effects on [CO 2 ] oS because pH oS >> pK, so that [HCO 3 − ] oS >> [CO 2 ] oS . In the case of HCO 3 − ( Figure 3B), CA has negligible effects on the transport rate, as described below. We would expect as much: because [HCO 3 − ] oS >> [CO 2 ] oS under physiological conditions, the CA-catalyzed reaction has little impact on [HCO 3 − ] oS . In the case of H + ( Figure 3C), the effect of CA on transport rate has, to our knowledge, not been tested. However, we suspect that the impact of CA might be muted, again because the high [HCO 3 − ] oS /[CO 2 ] oS ratio would tend to reduce the extent of the forward reaction in Figure 3C. Finally, in the case of CO 3 2− ( Figure 3D), CA has a negligible impact on the transport rate, as we discuss below. We would expect as much: because [HCO 3 − ] oS >>>> [CO 3 2− ] oS under physiological conditions, the rate of the reaction HCO 3 − → CO 3 2− + H + is apparently little influenced by CA-dependent changes in [H + ] oS .
Continuing the summary, the upper row of Figure 3 shows that, for fluxes of CO 2 ( Figure 3A) or HCO 3 − ( Figure 3B), CA magnifies pH changes in the nanodomain near the membrane. Conversely, the lower row of Figure 3 shows that, for fluxes of H + ( Figure 3C) or CO 3 2− ( Figure 3D), CA minimizes pH changes in the nanodomain near the membrane. Below, we show that it is possible to exploit the chemistry of reactions (2) and (3), as well as a variety of biophysical approaches, to identify the substrates carried by "HCO 3 − " transporters.

Carbonic Anhydrases and the Identification of Substrates of SLC4 Family Members
In theory, "HCO 3 − " transporters could carry any of the solutes involved in reactions (2) and (3). Distinguishing among them has been challenging because, in contrast with non-labile ions (e.g., sodium, potassium), the solutes of reactions (2) and (3) are interchangeable (i.e., they can be converted into each other), making direct measurements virtually impossible. Some investigators have resorted to surrogate substrates or kinetic approaches [60][61][62][63][64][65][66]. However, neither approaches are definitive: (i) No surrogate can mimic the real physicochemical properties of the substrates under consideration [67]. (ii) The only definitive conclusion that can come from kinetic approaches-and even then, only under favorable circumstances-is to rule out false hypotheses. Although kinetic studies can support a model, they can never rule one out. Thus, although various investigators may have had their hypotheses based on surrogate or kinetic data, none of these conclusions-by definition-could have been definitive.
An advancement towards solving this technical conundrum was the theoretical observation that the combination of CA inhibitors and their opposite effects on pH changes could help distinguish HCO 3 − vs. CO 3 2− transport across cell membranes [68][69][70]. However, early studies with CA inhibitors and pH measurements could not determine unambiguously the identity of the transported substrate because of limitations in the experimental system (native tissue), which almost certainly comprised an unknown mixture of acid-base transporters. Moreover, in these earlier studies, the investigators did not consider the possibility that "HCO 3 − " transporters could carry H + or CO 2 . Interestingly, preliminary work from our group suggests that the electrogenic Na/HCO 3 cotransporter-1 (variant A) can conduct CO 2 [71,72].
In the following sections, after a brief overview of the members of the SLC4 family of "HCO 3 − " transporters, we review a recent study from our group in which we were able to identify unambiguously the nature of the substrates carried by the "HCO 3 − " transporters of the SLC4 family.
Members of the SLC4 family are expressed throughout the body and are essential for regulating intracellular and whole-body pH, and for transporting acid-base equivalents across many epithelia. These proteins are implicated in a variety of diseases. For example, mutations of AE1 have been associated with type I distal renal tubular acidosis (RTA) and hereditary spherocytosis [85][86][87]. Mutations of NBCe1 have also been linked to type II proximal RTA, glaucoma, migraine, and suicidal ideation [88][89][90].
Many reviews on the SLC4 family are available, and we refer the interested reader to these for more details (refs. [91][92][93]). Figure 4 illustrates the three possible models of acid-base transport that we explored in our study [73]: HCO 3 − influx (panel A), CO 3 2− influx (panel B), and CO 2 /HCO 3 −stimulated H + efflux (panel C). For simplicity, we consider only the case in which a base enters the cell. Similar conclusions can be reached for the case in which a base exits the cell. We also omit the accompanying movements of Na + and/or Cl − and the corresponding postulated transporter stoichiometry of the members of the SLC4 that we studied.

Theoretical Role of Carbonic Anhydrase in Distinguishing Bicarbonate versus Carbonate versus Proton Transport across Cell Membranes
If the transporter mediates HCO 3 − entry into the cell ( Figure 4A), the result will be a decrease in [HCO 3 − ] oS . The lost HCO 3 − can be replenished by either diffusion (indicated by the dashed arrow), which does not affect pH oS or reaction (2). Because reaction (2) produces H + , it will cause a decrease in pH oS (see inset, lower left corner of panel A: solid black trace). Blocking CA (i.e., applying a CA inhibitor) will reduce H + production, thereby causing a smaller decrease in pH oS (dashed red trace).
If the transporter mediates CO 3 2− entry into the cell ( Figure 4B), the result will be a decrease in [CO 3 2− ] oS that triggers reaction (3), thereby replenishing CO 3 2− but also producing H + . The consequence is a decrease in pH oS (inset: solid black trace). Consuming some of this newly formed H + will be reaction (2) catalyzed by CA. Therefore, blocking CA will reduce H + consumption via reaction (2), causing a further decrease in pH oS (dashed red trace). Thus, the blockade of CA in model A vs. model B has opposite effects on pH oS .
If the transporter, stimulated by CO 2 or HCO 3 − , mediates H + efflux from the cell ( Figure 4C), the result will be an immediate increase in [H + ] oS , as reflected by a fall in pH os (inset: solid black trace). This triggers both H + diffusion away from the membrane and the CA-catalyzed reaction (2), both of which mitigate the rise in [H + ] oS . Blocking CA will slow H + consumption, magnifying the decrease in pH oS (dashed red trace).
versus Proton Transport across Cell Membranes Figure 4 illustrates the three possible models of acid-base transport that we explored in our study [73]: HCO3 − influx (panel A), CO3 2− influx (panel B), and CO2/HCO3 − -stimulated H + efflux (panel C). For simplicity, we consider only the case in which a base enters the cell. Similar conclusions can be reached for the case in which a base exits the cell. We also omit the accompanying movements of Na + and/or Cl  and the corresponding postulated transporter stoichiometry of the members of the SLC4 that we studied.  Comparing the hypothetical solid black vs. the dashed red traces in the insets of Figure 4A-C, we see that the effect of CA blockade on the direction of the pH oS change should, in principle, allows one to distinguish HCO 3 − transport on the one hand ( Figure 4A) from CO 3 2− or H + transport on the other ( Figure 4B,C).

Surface pH Studies Supporting Carbonate as the Substrate of NCBTs
In order to test the three possible models of transport in Figure 4, we co-expressed in oocytes (i) NBCe1-A ± CA IV as a test case for electrogenic NCBTs; (ii) AE1 ± CA IV as a test case for electroneutral AEs and (iii) NDCBE ± CA IV as an additional test case for electroneutral NCBTs. In all of these three cases, we exploited (i) the chemistry of reactions (2) and (3) at the outer surface of the plasma membrane, (ii) the CA inhibitor acetazolamide (ACZ), (iii) measurements of pH oS , (iv) heterologous expression of the proteins in Xenopus oocytes, and (v) mathematical simulations.
NBCe1-A appears to mediate the isodirectional movement of 1 Na + and 2 HCO 3 − ions (i.e., 1:2 stoichiometry), [93,94], as we could represent by doubling all stoichiometry values in Figure 4A. In principle, NBCe1-A could also move 1 Na + and 1 CO 3 2− in the same direction, as indicated by Figure 4B, or 1 Na + and 2 H + in the opposite direction in a CO 2 or HCO 3 − stimulated process, as we could represent by doubling all stoichiometry in Figure 4C. All three models are thermodynamically equivalent.
Because NBCe1-A is electrogenic, we simultaneously measured changes in NBC current (I NBC ) and pH oS with microelectrodes by shifting membrane voltage (V m ) using two-electrode voltage clamping technique. Details on this technical approach can be found in ref. [95]. Our data show that inhibition of CA IV by ACZ amplifies pH oS changes, thereby allowing us to rule out HCO 3 − transport (i.e., Figure 4A). In order to distinguish between the transport of 1 Na + and 1 CO 3 2− in the same direction vs. 1 Na + and 2 H + in opposite directions, we introduced a powerful new set of tools: we analyzed the amount of H + that appears at the cell surface (measured as a change in pH oS ) per electrical charge carried (e − , measured as change in I NBC ) in each model. Using the well-characterized H + channel H V 1 to calibrate our experimental data and a reaction-diffusion mathematical model that simulates our experiments with oocytes, we were able to predict the amount of H + /e − that appears at the cell surface. The mathematical model is an extension of the one employed in previous studies [56][57][58] and includes reactions (1) and (3) as well as the non-CO 2 /HCO 3 − buffers (i.e., HEPES in the extracellular space to mimic the composition of our perfusion solution and the intrinsic cytosolic buffers). Comparing the model prediction with our experimental data, we were able to rule out both the Na + + 2 HCO 3 − model (simulation predicted very low H + /e − ; see Figure 5A in Ref. [73]) and the Na + -2H + exchange model (simulation predicted very high H + /e − ; see Figure 5C in Ref. [73]). In fact, our data matched very closely the predictions for the transport of CO 3 2− (simulation predicted moderate H + /e − ; see Figure 5B in Ref. [73]). Thus, we definitively conclude that NBCe1-A cannot carry either HCO 3 − or H + and most likely carries 1 Na + and 1 CO 3 2− in the same direction. In the cases of the electroneutral transporters AE1 and NDCBE, we could not use the H + /e − approach. Instead, we acid loaded the cytoplasm of oocytes by exposure to CO 2 /HCO 3 − (the CO 2 enters the cell and generates HCO 3 − + H + ) and then used intracellular-pH microelectrodes to monitor the subsequent recovery (i.e., increase) of intracellular pH (pH i ), which reflects the activity of the transporter. In addition, we monitored pH oS changes during the rise in pH i .
Our data on AE1 show that inhibition of CA IV by ACZ reduces pH oS changes, thereby allowing us to rule out the transport of CO 3 2− ( Figure 4B) or H + ( Figure 4C) and confirm what investigators had long believed; namely, AE1 transports HCO 3 − ( Figure 4A). Our data on NDCBE show that inhibition of CA IV by ACZ amplifies pH oS changesthe opposite of what we observed with AE1-thereby allowing us to rule out HCO 3 − transport ( Figure 4A).
In summary, our study shows that NBCe1-A almost certainly carries CO 3 2− or a related substrate (e.g., the NaCO 3 − ion pair: NaHCO 3 − Na + + CO 3 2− ), AE1 does indeed carry HCO 3 − , and NDCBE does not carry HCO 3 − . We suggest that similar studies will likely show that all AEs transport HCO 3 − and all NCBTs transport CO 3 2− . Finally, our approach can also be used for clarifying the nature of the substrates of transporters (e.g., SLC26 transporters) and channels that traditionally have been thought to mediate the movement of "HCO 3 − ".

Relative Abundance of Members of the SLC4 and α-CA Families in Human Kidney
Before systematically examining the members of the SLC4 and α-CA families involved in renal HCO 3 − reabsorption, we used data from a recently published human study to obtain information on the relative abundance of mRNA and proteins in the kidney [96]. Although the transcriptome analysis is comprehensive and unbiased, the protein expression is relative to the tissue with the highest expression of that protein. Therefore, we cannot truly compare relative amounts of different proteins in the kidney. In some cases, mRNA and protein levels do not correlate well. The study's authors suggest that this discrepancy could be due to the specificity and affinity of different antibodies directed to different proteins, as well as the non-linearity of immunohistochemistry assays, or to post-translational modifications of proteins (e.g., secretion and proteolysis). We note that static mRNA levels do not need to correlate with static protein levels, let alone with the disposition of the protein (e.g., cellular localization, posttranslational modification) that is physiologically relevant. Thus, even though these data represent an impressive amount of work and may provide important insights for further experiments, one must interpret such data with caution.
The results of this study [96] are included in the Human Protein Atlas database (see "http://www.proteinatlas.org") (accessed on 20 January 2023).

SLC4 Family Members
Based on the RNAseq data of Fagerberg and coworkers [96], we classify the nine members of the SLC4 family that carry "HCO 3 − " into four major categories (very low, low, medium, and high), using the normalized tags per million (nTPM) value as an index of mRNA level (Table 1). For proteins, we simply use Fagerberg's categories (as low, medium, and high) to describe expression levels [96]. It is noteworthy-but see caveats above regarding protein values-that SLC4A4 (NBCe1) has both high mRNA and protein values. On the other hand, SLC4A7 (NBCn1) and SLC4A10 (NBCn2) both have very low or low mRNA levels but high protein values.
Below, we provide information on current knowledge of cellular and sub-cellular localization of members of this family along the nephron.

α-CA Family Members
For the 15 human α-CA family members (note: humans lack CA15), Table 2 summarizes the mRNA and protein expression levels following the same approach that we employed for the SLC4 family. We note that the CA2 and CA12 genes produce both the highest nTPM and protein values. On the other hand, CA4 has medium mRNA but low protein levels. Table 2. Classification of human α-CA family members in the kidney. mRNA and protein expression levels were classified based on the study [96]. Very low: ≤1 nTPM; low: 1 ≤ 15 nTPM; medium: 15 ≤ 50 nTPM; high: >50 nTPM. * N/A means either not tested or not detected. nTPM = normalized tags per million. Note that no data are available for CA15 because this gene has not been identified in humans [97].

Gene (Protein) Name mRNA Expression Protein Expression Very Low
Low Medium High Low Medium High N/A * These results are consistent with our current knowledge that CA II and CA XII are highly expressed in the kidneys but apparently are in conflict with the current impression that CA IV is similarly important. However, see the caveats above regarding reported protein levels. Note that CA IX overexpression has been associated with renal carcinoma [17].
Below, we provide information on current knowledge of cellular and sub-cellular localization of members of this family along the nephron.

Carbonic Anhydrases along the Nephron
The first report of CA activity in the kidneys dates back to 1941 when Davenport and Wilhelmi detected CA activity in the renal cortex of dogs, cats, and rats [98]. The identification of CA in the kidney was an important step toward understanding renal acid-base physiology.
The role of CA in urinary acidification emerged when investigators observed that the administration of CA inhibitors reduces the titratable acidity of the urine. Höber was the first to report that the addition of sulfanilamide in the perfusion fluid blocks urinary acidification in frog kidneys [99]. Studies by Pitts and Alexander also supported the role of CA in urinary acidification. These authors demonstrated that the two theories prevailing at the time on the mechanism of urinary acidification-reabsorption of bicarbonate vs. reabsorption of alkaline phosphate-could account only for a relatively small fraction of the maximum titratable acidity of the urine. Thus, they suggested that secretion of H + into the lumen was the only mechanism that could explain the amount of acid in the urine [100][101][102][103][104]. Pitts and Alexander correctly hypothesized that secretion of H + in renal tubules occurs in exchange for some filtered luminal cation, most likely Na + (as postulated earlier by Homer Smith [105,106]). Moreover, experiments with sulfonamide led these authors to suggest that the likely source of secreted H + was intracellular CO 2 hydration and that intracellular CA catalysis was an important part of this process [101]. During this time, the distal tubule was considered the main site of urinary acidification. Although some investigators had also suggested that the proximal tubule (PT) could play a role in urinary acidification [107][108][109], it was only in 1960 that Gottschalk and colleagues provided direct evidence on proximal acidification [110]. By performing microperfusion studies in rat kidneys, these authors observed that the fluid of the PT acidified to a pH of~6.8, consistent with the hypothesis that most bicarbonate reabsorption (>85%) occurs in this segment.
Micropuncture and microperfusion studies exploiting CA inhibitors demonstrate that HCO 3 − reabsorption in PTs (i) strongly depends on the presence of luminal/membraneassociated and cytosolic CA and (ii) occurs via H + secretion into the lumen and not by direct absorption of HCO 3 − across the apical membrane [111][112][113][114][115]. Burg and colleagues, working with isolated perfused tubules, demonstrated that the PT and thick ascending limb (TAL) are major nephron segments responsible for reabsorbing HCO 3 − and that they both rely on CA and a Na-coupled H + -secretion mechanism [116][117][118][119]. McKinney and Burg confirmed that the collecting duct contributes to urinary acidification by reabsorption of bicarbonate in a CA-dependent manner [116]. However, contrary to what had been believed, they found that H + secretion in this segment is independent of Na + transport [116].
Our current knowledge of renal CA and its localization along the nephron is the result of numerous biochemical, immunocytochemical, and histochemical studies [24,[120][121][122][123][124][125][126]. In 1975 Wistrand and coworkers, by employing affinity-chromatography techniques, were able to isolate CA from human kidneys and demonstrate that this CA was the same CA II of human RBCs [122]. These authors observed that a small percentage of the CA activity is not cytosolic but originates from a membrane-associated CA localized in both apical and basolateral membranes [127]. We now know that CA II, CA IV, and CA XII are the most prominent CA isozymes in the human kidney, with cytosolic CA II accounting for over 95% of the total renal CA activity and membrane-associated CA IV and CA XII accounting for the remaining 5% [24,111,120]. Although CA XIV is apparently not important in the human kidney, and the human genome lacks the CA15 gene, both of these membrane-associated CAs play a role in the rodent kidney [2,120].
Below we describe the mechanisms of HCO 3 − reabsorption along the nephron with special emphasis on our current knowledge about the localization and functional role of the three renal CA isozymes in humans. The same proteins or processes that mediate HCO 3 − reabsorption also generate "new HCO 3 − ". The only difference is that, in the case of HCO 3 − reabsorption, the H + secreted into the lumen titrates HCO 3 − , whereas in the case of new-HCO 3 − formation, the secreted H + titrates NH 3 (producing NH 4 + secretion) or buffers like phosphate and creatinine (producing titratable acidity).

Proximal Tubule
PTs are responsible for reabsorbing~80% to~85% of filtered HCO 3 − . Most of this HCO 3 − (after the luminal reaction HCO 3 − + H + → H 2 O + CO 2 ) moves in the form of CO 2 from the lumen, across the apical membrane (AM), and into the cytosol. After reconversion to HCO 3 − , the carbon crosses the basolateral membrane (BLM) in the form of CO 3 2− or HCO 3 − . Figure 5 illustrates the mechanisms of the preceding series of events. Mediating the majority of the H + secretion at the AM is Na-H exchanger 3 (NHE3), which uses the inward Na + gradient-established by the Na-K pump at the BLM-to exchange luminal Na + for intracellular H + [128][129][130]. Mediating a smaller fraction of H + secretion is the apical vacuolar-type H + pump (or V-type H + pump), which uses ATP hydrolysis to energize the extrusion of H + from the cell. A recent study on rat kidneys indicates that one variant of NBCn2 is present in the AM, where it could be responsible for the direct reabsorption of perhaps 20% of the reclaimed HCO 3 − [131].
were able to isolate CA from human kidneys and demonstrate that this CA was the same CA II of human RBCs [122]. These authors observed that a small percentage of the CA activity is not cytosolic but originates from a membrane-associated CA localized in both apical and basolateral membranes [127]. We now know that CA II, CA IV, and CA XII are the most prominent CA isozymes in the human kidney, with cytosolic CA II accounting for over 95% of the total renal CA activity and membrane-associated CA IV and CA XII accounting for the remaining 5% [24,111,120]. Although CA XIV is apparently not important in the human kidney, and the human genome lacks the CA15 gene, both of these membrane-associated CAs play a role in the rodent kidney [2,120]. Below we describe the mechanisms of HCO3 − reabsorption along the nephron with special emphasis on our current knowledge about the localization and functional role of the three renal CA isozymes in humans. The same proteins or processes that mediate HCO3 − reabsorption also generate "new HCO3 − ". The only difference is that, in the case of HCO3 − reabsorption, the H + secreted into the lumen titrates HCO3 − , whereas in the case of new-HCO3 − formation, the secreted H + titrates NH3 (producing NH4 + secretion) or buffers like phosphate and creatinine (producing titratable acidity).

Proximal Tubule
PTs are responsible for reabsorbing 80% to 85% of filtered HCO3 − . Most of this HCO3 − (after the luminal reaction HCO3 − + H +  H2O + CO2) moves in the form of CO2 from the lumen, across the apical membrane (AM), and into the cytosol. After reconversion to HCO3 − , the carbon crosses the basolateral membrane (BLM) in the form of CO3 2− or HCO3 − . Figure 5 illustrates the mechanisms of the preceding series of events. Mediating the majority of the H + secretion at the AM is Na-H exchanger 3 (NHE3), which uses the inward Na + gradient-established by the Na-K pump at the BLM-to exchange luminal Na + for intracellular H + [128][129][130]. Mediating a smaller fraction of H + secretion is the apical vacuolar-type H + pump (or V-type H + pump), which uses ATP hydrolysis to energize the extrusion of H + from the cell. A recent study on rat kidneys indicates that one variant of NBCn2 is present in the AM, where it could be responsible for the direct reabsorption of perhaps 20% of the reclaimed HCO3 − [131]. In the lumen, secreted H + combines with filtered HCO 3 − , thereby producing CO 2 and H 2 O under the catalytic action of CA IV. The newly formed CO 2 and H 2 O cross the AM mostly through the water channel aquaporin AQP1 [132][133][134][135]. In the cytosol, CA II promotes rapid conversion of CO 2 and H 2 O into HCO 3 − and the first of two H + . This newly formed H + then recycles back into the lumen, whereas the newly produced HCO 3 − will further dissociate to form a second H + plus CO 3 2− and exit the cell across the BLM in the form of CO 3 2− via NBCe1-A [136]. This second H + also recycles back into the lumen. Because the debate continues as to whether, in PT cells, NBCe1-A truly operates with a 1Na + :3"HCO 3 − " stoichiometry or, as in most other cells, with a 1Na + :2"HCO 3 − " stoichiometry, in Figure 5 we show a single CO 3 2− only (i.e., consistent with a 1:2 stoichiometry). An apparent 1:3 stoichiometry could be explained, for example, with the exit of an additional HCO 3 − ion via NBCe1-A. Alternatively, the apparent necessity to invoke a 1:3 stoichiometry could be the result of measurement resolution-that is, using available macroscopic measurements of [Na + ] and ["HCO 3 − "] near the inner and outer sides of the membrane rather than the relevant but unavailable measurements in the nanodomains adjacent to NBCe1-A.

Subcellular Localizations of CA Isozymes
CA II is present in the cytosol, and the glycosylphosphatidylinositol (GPI)-linked CA IV is expressed on both AM and BLM (less on the BLM) of the S1 and S2 segments of the PT [120,141]. CA XII is expressed only on the BLM [9].
Regarding RPTPγ, previous studies from our group suggest that the extracellular CA-like domain (CALD) senses CO 2 or HCO 3 − and the phosphatase domain turns on a downstream signaling cascade that reaches key acid-base transporters [137,138,140]. Interestingly, the CALD of RPTPγ, compared to CA II, lacks several key residues necessary for catalysis, including two of the three histidine residues essential for coordinating Zn 2+ . If a physiological role of RPTPγ is indeed to sense basolateral CO 2 and HCO 3 − , one would expect it to lack catalytic activity (i.e., the molecule should not interconvert the solutes that it is detecting), as is indeed the case [142,143].

Functional Interactions with Acid-Base Transporters
As illustrated in Figure 5, the role of apical CA IV in HCO 3 − reabsorption is to convert luminal HCO 3 − to CO 2 for uptake across the AM. The role of cytosolic CA II is to convert the CO 2 entering across the AM to HCO 3 − and H + . Thus, both CA IV and CA II enhance the gradient that favors CO 2 uptake across the AM into the PT cell. CA II also provides cytosolic H + for extrusion by NHE3 and V-type H + pump across the AM, and HCO 3 − (which generates CO 3 2− ) for transport by NBCe1-A across the BLM. One group, following the lead of others working on AE1 in RBCs, reports that CA II binds to the C-terminus of NBCe1-A, creating a transport metabolon that enhances NBCe1-A activity [144,145]. In addition, others have reported that CA IV binds to the 4th extracellular loop of NBCe1-A, also stimulating NBCe1-A activity [146]. These papers are consistent with the idea that cytosolic CA II provides the substrate for NBCe1-A (HCO 3 − or CO 3 2− ), while CA IV at the outer surface of the BLM dissipates the substrate [146]. However, our group detected neither binding of CA II to NBCe1-A, nor acceleration of transport, even after fusing CA II to the C-terminus of NBCe1-A [147][148][149]. Moreover, our recent work (pH S experiments of Figure 2 in ref. [73]) shows that CA IV activity-±expression or ±ACZ-does not affect the NBCe1-A current (and therefore transport) induced by identical electrical driving forces [73]. As noted in our discussion of Figure 3, if HCO 3 − or CO 3 2− is the transported ion, little is gained by interconverting HCO 3 − and CO 2 . On the other hand, if CO 2 is the transported species, this interconversion has major effects on enhancing diffusion.
Here, in light of our findings that NBCe1-A transports CO 3 2− , we suggest that the role of CA IV at the BLM is to minimize local basolateral pH changes caused by CO 3 2− transport. Figure 6 shows the effects of HCO 3 − vs. CO 3 2− efflux on pH oS in the presence (solid black trace) and absence (dashed red trace) of CA IV. If NBCe1-A were to transport HCO 3 − , then CA IV would accentuate the alkalinity of the extracellular surface. If NBCe1-A were to transport CO 3 2− , CA IV would reduce the alkalinity. In preliminary work, we increased peritubular [K + ] to depolarize mouse PTs (thereby driving Na + and "HCO 3 − " into the PT cell) and observed the expected decrease in pH oS . Performing this maneuver in the presence of a peritubular CA inhibitor markedly increased the magnitude of the pH oS decrease. These observations are consistent with the CO 3 2− transport model ( Figure 6B). However, because PTs express many acid-base transporters, further investigations-for example, the use of PTs from knockout (KO) mice lacking specific acid-base transporters-are needed.
of CA IV at the BLM is to minimize local basolateral pH changes caused by CO3 2− transport. Figure 6 shows the effects of HCO3 − vs. CO3 2− efflux on pHoS in the presence (solid black trace) and absence (dashed red trace) of CA IV. If NBCe1-A were to transport HCO3 − , then CA IV would accentuate the alkalinity of the extracellular surface. If NBCe1-A were to transport CO3 2− , CA IV would reduce the alkalinity. In preliminary work, we increased peritubular [K + ] to depolarize mouse PTs (thereby driving Na + and "HCO3 − " into the PT cell) and observed the expected decrease in pHoS. Performing this maneuver in the presence of a peritubular CA inhibitor markedly increased the magnitude of the pHoS decrease. These observations are consistent with the CO3 2− transport model ( Figure  6B). However, because PTs express many acid-base transporters, further investigationsfor example, the use of PTs from knockout (KO) mice lacking specific acid-base transporters-are needed. Regarding a potential physiological role for CA XII at the BLM, we suggest that this enzyme, as CA IV, minimizes pHoS changes at the BLM.
To summarize the actions of CAs in the PT, the apical CA IV and cytosolic CA II are complementary in accelerating the transmembrane CO2 flux [56][57][58]. By converting CO2 to the far-more-abundant HCO3 − , CA II also accelerates the flux of "carbon" from the apical to the basolateral membrane. In the process, CA II also provides cytosolic substrates for apical H + extrusion and basolateral "HCO3 − " efflux. The CA IV and XII at the BLM, however, do not play a substantial (or measurable) role in dissipating the "product" of NBCe1-A activity (i.e., the product being the appearance of CO3 2− in the extracellular nanodomain near NBCe1-A). Instead, these CAs stabilize pH in this nanodomain. Regarding a potential physiological role for CA XII at the BLM, we suggest that this enzyme, as CA IV, minimizes pH oS changes at the BLM.
To summarize the actions of CAs in the PT, the apical CA IV and cytosolic CA II are complementary in accelerating the transmembrane CO 2 flux [56][57][58]. By converting CO 2 to the far-more-abundant HCO 3 − , CA II also accelerates the flux of "carbon" from the apical to the basolateral membrane. In the process, CA II also provides cytosolic substrates for apical H + extrusion and basolateral "HCO 3 − " efflux. The CA IV and XII at the BLM, however, do not play a substantial (or measurable) role in dissipating the "product" of NBCe1-A activity (i.e., the product being the appearance of CO 3 2− in the extracellular nanodomain near NBCe1-A). Instead, these CAs stabilize pH in this nanodomain.

Thick Ascending Limb
Approximately 10-15% of filtered HCO 3 − is reabsorbed in the TAL. The mechanism of HCO 3 − reabsorption in the TAL (Figure 7) is very similar to that in the S1 and S2 segments of the PT ( Figure 5). In the TAL, NHE3/NHE2 and the V-type H + pump extrude H + across the AM [150]. In the lumen, the secreted H + combines with HCO 3 − , producing CO 2 and H 2 O. Because the AM of the TAL is tight to NH 3 and water and, to date, no CO 2 -conducting membrane proteins (e.g., AQPs) have been identified at the AM, the newly formed CO 2 enters the cell likely via diffusion only. Apical CA IV and cytosolic CA II, by maximizing the transmembrane CO 2 gradient across the AM, enhance CO 2 influx across the AM. Once inside the cytosol, the CO 2 combines with H 2 O to form HCO 3 − and H + . In contrast to PTs, where NBCe1-A exports Na + and CO 3 2− across the BLM, here in the TAL, AE2 exchanges cytosolic HCO 3 − for Cl − at the BLM. In addition to AE2, investigators have identified NBCn1 and (in rat) NBCn2 in the BLM of the TAL [131,[151][152][153]. Because NBCn1 and NBCn2 normally operate in an inward direction [154], it seems unlikely that they contribute to HCO 3 − reabsorption per se. Instead, their roles may be to "facilitate" transcellular NH 4 + transport from the TAL to the collecting duct [155,156].
the AM. Once inside the cytosol, the CO2 combines with H2O to form HCO3 − and H + . In contrast to PTs, where NBCe1-A exports Na + and CO3 2− across the BLM, here in the TAL, AE2 exchanges cytosolic HCO3 − for Cl − at the BLM. In addition to AE2, investigators have identified NBCn1 and (in rat) NBCn2 in the BLM of the TAL [131,[151][152][153]. Because NBCn1 and NBCn2 normally operate in an inward direction [154], it seems unlikely that they contribute to HCO3 − reabsorption per se. Instead, their roles may be to "facilitate" transcellular NH4 + transport from the TAL to the collecting duct [155,156]. The TAL reabsorbs luminal NH4 + via the Na-K-Cl cotransporter 2 (NKCC2) and the renal outer medullary K + channel (ROMK) [157,158]. Once inside the cell, NH4 + dissociates into NH3 and H + . It has been suggested that NBCn1 and NBCn2 neutralize this H + , thereby enhancing the formation of intracellular NH3 and promoting NH3 diffusion through the cell to the BLM. There, the NH3 exits the TAL cell, possibly via AQP1 [159], diffuses through the interstitial fluid, and finally crosses the BLM of the -intercalated cell via RhBG and RhCG [155,156,160]. Consistent with this hypothesis, in vivo and in vitro studies show that metabolic acidosis (MAc) increases NBCn1 expression [131,155,161].
Note that in Figure 7 we illustrate NBCn1 and NBCn2 as moving CO3 2− (rather than HCO3 − ) into the cell, consistent with preliminary data from our group. However, because this transporter is electroneutral, in Figure 7 we tentatively illustrate the cotransport of 2 Na + ions. Additional work is needed to elucidate the stoichiometry of NBCn1 and NBCn2.  The TAL reabsorbs luminal NH 4 + via the Na-K-Cl cotransporter 2 (NKCC2) and the renal outer medullary K + channel (ROMK) [157,158]. Once inside the cell, NH 4 + dissociates into NH 3 and H + . It has been suggested that NBCn1 and NBCn2 neutralize this H + , thereby enhancing the formation of intracellular NH 3 and promoting NH 3 diffusion through the cell to the BLM. There, the NH 3 exits the TAL cell, possibly via AQP1 [159], diffuses through the interstitial fluid, and finally crosses the BLM of the α-intercalated cell via RhBG and RhCG [155,156,160]. Consistent with this hypothesis, in vivo and in vitro studies show that metabolic acidosis (MAc) increases NBCn1 expression [131,155,161].
Note that in Figure 7 we illustrate NBCn1 and NBCn2 as moving CO 3 2− (rather than HCO 3 − ) into the cell, consistent with preliminary data from our group. However, because this transporter is electroneutral, in Figure 7 we tentatively illustrate the cotransport of 2 Na + ions. Additional work is needed to elucidate the stoichiometry of NBCn1 and NBCn2.

Subcellular Localizations of CA Isozymes
CA II is present in the cytosol of virtually all renal cells except for those in the tip of Henle's loop and the thin ascending limb [120]. Within Henle's loop, CA IV and CA XII are present only in the TAL, with similar localization as in the PT (i.e., CA IV is in both the AM and BLM and CA XII in the BLM.

Functional Interactions with Acid-Base Transporters
As illustrated in Figure 7, the role of apical CA IV in HCO 3 − reabsorption is to convert luminal HCO 3 − to CO 2 for uptake across the AM. The role of cytosolic CA II is to promote the consumption of incoming CO 2 , thereby accelerating CO 2 influx, and to provide cytosolic H + for extrusion by NHE3/NHE2 and the V-type H + pump across the AM, and HCO 3 − for export by AE2 across the BLM. These roles of apical CA IV and cytosolic CA II are similar to those discussed above for the PT.
Some investigators have reported that CA II and AEs can physically interact, thereby stimulating AEs activity [162][163][164][165]. According to other reports, CA IV binds to the 4th extracellular loop of AE1 (probably also AE2 and AE3), also stimulating AE1 activity [166]. However, other studies have shown no evidence of direct binding of CA II to the C-terminus of AE1 [147,167].
The work of Piermarini and colleagues [147] confirmed that liquid-phase AE1-C terminus (Ct) can bind to solid-phase CA II when-as in the earlier work-they fused the AE1-Ct to GST (which, significantly, forms dimers). Piermarini et al. replicated these results for GST-NBCe1-Ct and GST-NDCBE-Ct (collectively SLC4-Ct). However, when they reversed the orientation and applied liquid-phase CA II to solid-phase GST-SLC4-Ct, they observed no binding. Even when CA II was in the solid phase, SLC4-Ct failed to bind in the absence of GST. Thus, the interaction between the Ct of AE1 (and also of NBCe1 and NDCBE) does indeed occur but is not physiologically relevant because it requires that (i) the Ct be fused to GST and (ii) the GST fusion protein be liquid phase.
Moreover, our recent work ( Figure 6 and Supplemental Table 3A in ref. [73]) shows that in oocytes expressing AE1 (±CA IV or ±ACZ) and exposed to Cl − -free CO 2 /HCO 3 − solution, the presence of CA IV does not affect the rates of pH i recovery [73]. These experiments follow the trans-side rule of Musa-Aziz et al. [56][57][58]: it is legitimate to use pH measurements to assess the impact of a CA on the activity of an acid-base transporter only if the CA and the pH probe are on opposite sides of the membrane (e.g., CA IV on the outside, pH on the inside). The earlier authors added the CA II to the cytosol and measured pH on the same side. In their experiments, they saw that CA II markedly accelerated the AE1-dependent pH i changes. This would have occurred regardless of the effect (if any) of CA II on the transport rate because CA II-interposed between AE1 and the pH i probe-was responsible for translating the HCO 3 − flux into a pH i signal. Aside from the experimental data, as noted above in our discussion of Figure 3 as well as in the above corresponding section for PT, if HCO 3 − or CO 3 2− is the transported ion, the advantage of a CA-catalyzed interconversion of HCO 3 − and CO 2 for "HCO 3 − " transport would be small.
Here, in light of our findings that AE1 transports HCO 3 − and that NBCn1 and NBCn2 appear to carry CO 3 2− we propose that the role of CA IV at the BLM is not related to AE2 but to NBCn1 and NBCn2. Thus, we suggest that the CA IV (and possibly CA XII) minimizes pH oS changes at the BLM caused by CO 3 2− entry by NBCn1 and NBCn2 (see insert, lower left corner of panel B of Figure 4: solid black trace). This pH oS stabilization would presumably be beneficial to other nearby pH-sensitive proteins.

Tubules Distal to the Thick Ascending Limb
We have seen that most HCO 3 − reabsorption occurs in the PTs (~80-85%) and the TAL (~10-15%). The remaining~5-10% of HCO 3 − reabsorption occurs in the distal nephron (i.e., from the macula densa, which marks the beginning of the distal convoluted tubule through the inner medullary collecting duct).
Like the tubule segments from the beginning of the PT to the end of the TAL (which expresses NKCC2), the earliest tubule segment after the macula densa, the distal convoluted tubule 1 (DCT1), has one cell type. This DCT1 cell (which expresses the Na/Cl cotransporter NCC) does not appear to participate in acid-base transport. The next segment, the DCT2, has two cell types, DCT2 cells (with NCC + epithelial Na + channel ENaC) and intercalated cells (ICs), the latter of which do participate in acid-base transport. The third segment, the connecting tubule (CNT), has both CNT cells (ENaC + AQP2) and ICs. The fourth segment, the initial collecting tubule (ICT), also has two cell types, the segment-specific principal cells (PCs; ENaC + AQP2) and ICs. The fifth segment, which begins after the first confluence of ICTs, is identical to the ICT but has a new name, the cortical collecting tubule (CCT) or duct; it has both segment-specific PCs as well as ICs. The sixth post-macula-densa segment is the outer medullary collecting duct (OMCD), which again has segment-specific PCs as well as ICs. Finally, the inner medullary collecting duct (IMCD) has ICs in its initial part, but thereafter IMCD cells (AQP2 + UT-A1) [168][169][170][171][172][173][174]. The proportion of PCs versus ICs varies along the segments of the distal nephron, among and within species, and with physiological conditions [170,172,175].
Among the cell type listed above, only the ICs perform substantial transepithelial acidbase transport, with perhaps some contribution from the IMCD cells. The ICs comprise three subtypes: type-A or α-ICs (dominant in OMCD), type-B or β-ICs (dominant in CNT through CCT), and non-A/non-B [172,175,176].
The α-ICs secrete H + into the tubule lumen and thus are responsible for reabsorbing the remaining 5-10% of filtered HCO 3 − that enters the distal nephron ( Figure 8). These cells perform this task by secreting H + into the lumen via the V-type H + pump and the H + /K + pump, both on the AM [176][177][178]. CA IV at the AM converts filtered luminal HCO 3 − and secreted H + to CO 2 and H 2 O. CO 2 presumably enters the cell via the Rh proteins RhBG and RhCG [160]. In the cytosol, CA II catalyzes the conversion of the incoming CO 2 and H 2 O to HCO 3 − and H + . The newly formed HCO 3 − exits the BLM through the renal variant of AE1 [176][177][178]. Note that the roles of apical CA IV and cytosolic CA II in the α-IC are similar to those discussed above for the PT and TAL cells.
ing tubule (CCT) or duct; it has both segment-specific PCs as well as ICs. The sixth postmacula-densa segment is the outer medullary collecting duct (OMCD), which again has segment-specific PCs as well as ICs. Finally, the inner medullary collecting duct (IMCD) has ICs in its initial part, but thereafter IMCD cells (AQP2 + UT-A1) [168][169][170][171][172][173][174]. The proportion of PCs versus ICs varies along the segments of the distal nephron, among and within species, and with physiological conditions [170,172,175].
Among the cell type listed above, only the ICs perform substantial transepithelial acid-base transport, with perhaps some contribution from the IMCD cells. The ICs comprise three subtypes: type-A or -ICs (dominant in OMCD), type-B or β-ICs (dominant in CNT through CCT), and non-A/non-B [172,175,176].
The -ICs secrete H + into the tubule lumen and thus are responsible for reabsorbing the remaining 5-10% of filtered HCO3 − that enters the distal nephron ( Figure 8). These cells perform this task by secreting H + into the lumen via the V-type H + pump and the H + /K + pump, both on the AM [176][177][178]. CA IV at the AM converts filtered luminal HCO3 − and secreted H + to CO2 and H2O. CO2 presumably enters the cell via the Rh proteins RhBG and RhCG [160]. In the cytosol, CA II catalyzes the conversion of the incoming CO2 and H2O to HCO3 − and H + . The newly formed HCO3 − exits the BLM through the renal variant of AE1 [176][177][178]. Note that the roles of apical CA IV and cytosolic CA II in the -IC are similar to those discussed above for the PT and TAL cells.  The β-ICs cells secrete HCO 3 − into the tubule lumen [76,179]. Opposite to the α-ICs, the β-ICs express the V-type H + pump in the BLM, and a Cl-HCO 3 exchanger in the AM. However, this apical Cl-HCO 3 exchanger is pendrin (SLC26A4).
Non-A/non-B ICs, because they express both the V-type H + pump and pendrin in the AM [169,170], are intermediate between α-ICs and β-ICs (or, some say, a hybrid of the two), perhaps representing an intermediate state of cell differentiation [180]. For example, Purkerson and colleagues observed that MAc causes β-cells to become α-cells [181]. This transformation requires a large extracellular-matrix protein called hensin (from Japanese henshin, transformation) or deleted in malignant brain tumors 1 (DMBT1), as well as other proteins [180,182]. Indeed, mice without DMBT1 in the ICs have only a β-IC-like phenotype and develop MAc [183]. Inducing chronic MAc in rats leads to increased number of α-ICs. Similarly, inducing chronic metabolic alkalosis (MAlk) increases the number of β-ICs [180,184,185].
A recent study shows that the genetic deletion of NBCe1 (either global or PT-specific KOs) eliminates the typical IC response to MAc (e.g., an increase in α-IC and decrease in β-ICs) and is consistent with the idea of a link between PTs and ICs [186].

Subcellular Localizations of CA Isozymes
High amount of CA II is present in the cytosol of all ICs in the distal nephron. CA IV is expressed only in the AM of α-ICs [120,126]. Principal cells also have CA II, but the amount is significantly lower than in the ICs [187].

Functional Interactions with Acid-Base Transporters
The sub-cellular localization of CAs, as well as the presence of a H + -secretory mechanism at the AM and a HCO 3 − -exit mechanism at the BLM of α-ICs, provide all the tools necessary for these cells to perform H + secretion in conjunction with HCO 3 − reabsorption. Under normal physiological conditions, the contribution of ICs to whole-kidney HCO 3 − reabsorption is relatively small. However, these cells may play a critical role as they "fine tune" urinary acid secretion. Moreover, it appears that these cells can compensate for impaired HCO 3 − reabsorption in the preceding tubules [188]. A relatively low abundance of NCBTs (i.e., known or putative CO 3 2− transporters) on the BLM of ICs correlates with a relatively low expression of membrane-associated CAs at the BLM. This is consistent with our hypothesis that the role of membrane-associated CAs is not so much as to provide substrates to "HCO 3 − " transporters as to stabilize pH in the nanodomains near H + and CO 3 2− transporters. It will be informative to re-examine these issues with the accumulation of future data on the expression of NCBTs and CAs in the distal nephron.

Conclusions
CA activity has long been recognized as critically important for normal physiological function. For this reason, since the discovery of the first CA, investigators have focused considerable attention on the identification of CAs in a wide range of cells, how these CAs respond to physiological and pathophysiological challenges, and-using tools of molecular biophysics-the mechanisms of action of the CAs. Because of the fundamental role of CAs in acid-base homeostasis, some of these studies have focused on the interactions of CAs with acid-base transporters. As illustrated for the PT, TAL, and α-IC, the role of apical CA IV and cytosolic CA II is to accelerate CO 2 influx across the AM, to speed "carbon" diffusion from AM to BLM, and to provide H + and HCO 3 − as substrates for apical H + extrusion and basolateral "HCO 3 − " transport via members of the SLC4 family. Whereas the role of apical CA IV is to accelerate CO 2 influx across apical membranes (i.e., PT, TAL, and α-IC), the role of basolateral CA IV in the renal PT is to provide the H + necessary to titrate the newly transported CO 3 2− to HCO 3 − . By preventing an excessive rise in pH oS , basolateral CA IV (and CA XII) protects pH-sensitive processes near NBCe1-A at the outer surface of the BLM. We suggest that it is likely that membrane-associated CAs play similar buffering roles when coupled with other members of the SLC4 that carry CO 3 2− . These membrane-associated CAs would similarly minimize pH oS changes near the H + -extrusion mechanism but not near HCO 3 − transporters (where they would accentuate pH oS changes). In the case of αand β-ICs, the interconversion from one subtype to the other must involve substantial changes in the expression of CA IV, changes that coordinate with those in the acid-base transporters.